4990 12, 27 (1947); L. D. Wright, H. R. Skeggs. and E. L. Cresson, Proc. SOC. Exp. Bo/. Med., 64, 150 (1947); N. M. Green, Biochem. J., 89, 599 (1963). (32) R. E. Rosenfield, Jr.. and R. Parthasarathy, J. Am. Chem. SOC.,96, 1925 (1974).
(33) D. B. Boyd, J. Phys. Chem., 78, 1554 (1974); ibid., 78,2604(1974): Theor. Chim. Acta, 30, 137 (1973), and personal communication to one of us (R.P.). (34) H.E. Van Wart, L. L. Shipman. and J. A. Scheraga, J. Chem. phys., in press.
Mechanism of Phosphorylation by N'-Phosphorocreatine. Concurrent Formation of Creatine and Creatinine' ,2 Gary W. Allen and Paul Haake* Contribution f r o m the Department ojchemistry, Wesleyan Uniuersity, Middletown, Connecticut 06457. Receiued Nouember 24, I975
Abstract: The mechanism of phosphorylation by phosphorocreatine and the mechanism of creatinine formation have been investigated in aqueous solution. The hydrolysis of phosphorocreatine (PC) was found to occur via two pathways: pathway A leads to the production of creatine and inorganic phosphate; pathway B leads to the production of creatinine and inorganic phosphate. At pH values above 1.0, pathway A accounts for greater than 90% of the hydrolysis; pathway B becomes predominant only in strongly acidic solutions. For pathway A, the bell-shaped, pH-rate profile with a rate maximum at pH 1-2 (k,,, = 1.77 X min-I), AS* = -2 eu, and k(HzO)/k(D*O) = 0.86 strongly support a metaphosphate mechanism for phosphorylations by phosphorocreatine. The biological implications with regard to the action of ATP:creatine N-phosphotransferase (creatine kinase, E.C. 2.7.3.2) and the origin of urinary creatinine are discussed.
N'-Phosphorocreatine (1, PC), a phosphorog~anidine,~ plays an essential role by phosphorylating ADP (eq 1) during periods of rapid utilization of ATP in muscle. Our studies of the mechanism of phosphorylation by the simple phosphoroguanidines, 2 and 3, appears to involve proton transfer leading to unimolecular cleavage to the protonated guanidine and the reactive intermediate, monomeric metaphosphate ion, P03-,234 which is the actual phosphorylating agent. Phosphoroguanidines appear to be the most reactive precursors of metaphoshate.^ The presence of the carboxylate group in PC presents possibilities for functional group and ionization effects which could lead to special reactivity in this natural phosphorylating agent. Therefore, in order to assess the significance of the metaphosphate mechanism with respect to the in vivo phosphorylation reaction involving PC, the in vitro mechanism of phosphorylation of water (hydrolysis) by PC has been studied. This paper is addressed to the chemistry of hydrolysis of 1, the implications for biological phosphorylation by 1, and the mechanism of formation of creatinine ( 5 ) .
m HOPNHzC II 4 + \ I-0 0
HzN+Nyo /N H3C
/NR H3C
1,R = CH,CO,H (W-phosphorocreatine) 2, R = CH, 3, R = CH,C,H, 1
+ ADP a ATP + H,N-C
5 (creatinine)
PHz +
(1)
'NCH,co2-
I
CH3 4 (creatine)
Previous investigations on PC are inconclusive. PC has been shown to be stable in alkaline solutionS but to undergo rapid hydrolysis in acid with a rate maximum a t p H 1-3.6v7 It has also been reported that PC hydrolyzes in acid some 30 times faster than an unsubstituted phosphoroguanidine,s therefore, Journal of the American Chemical Society
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indicating possible intramolecular catalysis by the carboxylate group. The products of hydrolysis of PC are reported to be creatine (4) and a small amount of creatinine ( 5 ) . 9A mechanism for formation of 5 involving cyclization to 6 followed by P-N cleavage and ring opening (eq 2) has been proposed;I0 evidence for this mechanism includes the report of an induction period." However, hydrolysis in I80-enriched water has demonstrated that this mechanism cannot be correct since no label was found in the creatine isolated from the reaction.'* Recently, it has also been shown that 5 hydrolyzes to creatine more slowly than 1 does.'!
I
CH3 6
Results Potentiometric Titration. A potentiometric titration was carried out on the disodium salt of PC under the same conditions as those employed in the kinetic studies above p H 0.78, that is, T = 30.5 OC and p = 0.20 N in added NaCl. The overlapping dissociation constants were calculated by the method of Noyes.I4 The dissociation constants (Table I) are assigned in eq 3; the bases for the assignments have been disc~ssed.~.'~ Products of the Hydrolysis of PC. The rates of hydrolysis of PC were measured a t 30.47 f 0.05 O C in the p H range of 0.78-5.58 in buffered solutions with the ionic strength maintained constant a t 0.20 N by the addition of NaCl or NaC104, and in 0.40-4.00 N HC104. On hydrolysis, PC produces inorganic phosphate and both 4 and 5. Both products were detected by paper chromatography for the hydrolysis of PC in 1 .O N HC104. The Jaff6-Folin determination16 demonstrated conclusively that 5 is ca. 5% of the products of the hydrolysis of PC a t p H 5.22 and 5.88. A quantitative measure of the product composition is afforded by the absorbance a t 225.0 nm a t the end of the hy-
August 4, 1976
499 1
5600 140
5200 I20
4 4800
I
v
6
100
-ia 8
1
cu
4400
80 4000 60
3600 I .oo
0
2.00
3.00
5.00
4.00
PH
Figure 1. The
molar absorptivities ( e ) of creatinine ( 5 ) and creatine (4) at 225.0 n m as a function of pH: 0, creatinine; A, creatine
0.80 0.80
a
0.60
:E 0.60
I
6
ps
.-a-
t a 0.40 3 z
V
C c
t
--::
0.40
t
-u
s
0.20
0.20
0
1.00
2.00 CHCI0,l
3.00
4.00
(M)
Figure 2. The mole fraction of creatinine produced by hydrolysis of PC at 30.5 "C ( N = 4.00 N) as a function of the concentration of HC104. The curve was calculated from the constants (at the appropriate ionic strength) listed in Tables I I and I l l . Table
I. pK, Values for the Ionization of Phosphorocreatine (1) Dissociable group
First H of P moiety (pK,I) -COOH (pK,II) Second H of P moiety (pKa1l1) Guanidine (DK,~")
PKa' -0.41 2.84 4.46
0
1.00
2.00
3.00
4.00
5.00
PH Figure 3. The mole fraction of creatinine produced by hydrolysis of PC at 30.5 "C as a function of pH: 0.1= 0.20 N; A, = 4.00 N . The curves were calculated from the constants (at the appropriate ionic strength) in Tables I I and 111.
PKa O (4: 1 :I :2). Creatine and creatinine were detected by spraying the dried chromatograms with alkaline ferricyanide-nitroprusside reagent.32 The following R,- values and behavior to this reagent were observed: creatine, Rf = 0.34, orange; creatinine, Rf = 0.55, orange, turning blue after a few minutes. For both compounds the detection limit was 1 Pg. Product Identification. The hydrolysis of PC was carried out at 5.0 M and 1.0 M HC104 (PC concentration = 0.1 M) and at pH 3.61 (0.10 M acetate, P C concentration = 0.05 M) at 30.5 f 0.1 OC under the same conditions used for the kinetic runs. After I O half-lives ten aliquots of each solution were applied to the chromatography paper and the chromatograms developed as described above. In 4.0 M HC104 the only guanidine-containing product detected was creatinine. At 1.0 M HC104 both creatine and creatinine were detected. At pH 3.61 only creatine was detected. Jaffi-Folin Creatinine Determination.I6 A 2.00-ml aliquot of the solution to be analyzed, containing up to 5.0 X M creatinine, was added to 3.00 ml of a freshly prepared alkaline picrate solution (25 ml of a saturated picric acid solution and 5 ml of a 10%N a O H solution) in a test tube. After allowing 10 min for color development, 3 ml of the solution was added to a 1-cm cuvette and the absorbance a 525 nm was read against a blank. The procedure was calibrated with standard solutions of creatinine, and the absorbance a t 525 nm was found to vary linearly with creatinine concentration up to 5.0 X M. The procedure was found to be reproducible to within 5%. When this procedure was used to determine creatinine in the presence of large amounts of creatine, a correction for the slow conversion of creatine into creatinine, catalyzed by the alkaline picrate solution, had to be applied.
+
Acknowledgment. After this research was well advanced, we were made aware of a related thesis by Dr. A. R. Macrae (Cambridge, 1964) from which Professor V. M. Clark graciously sent us a copy of the relevant sections.
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References and Notes (1) This research was supported by Grant AN12743 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. (2) A preliminary report of a portion of this research has appeared: P. Haake andG. W. Allen, Proc. NatLAcad. Sci. U.S.A.,68, 2691 (1971);seealso G. W. Allen, Ph.D. Thesis, Wesleyan University, 1971. (3) Nomenclature: PC has been commonly called "creatine phosphate" which is unfortunate since that implies a salt; M-phosphorocreatine correctly indicates a P-N covalent bond. (4) P. Haake and G. W. Alien, J. Am. Chem. Soc., 95, 8080 (1973). (5) K. Zeile and G. Fawaz, Hoppe-Seyler's Z. Physiol. Chem., 256, 193 (1938). (6) C. H. Fiske and Y. Subbarow, J. Biol. Chem., 81, 629 (1929). (7) K. Lohmann, Biochem. Z.. 194, 306 (1938). (8) G. Fawaz and K. Zeile, Hoppe-Seyler's 2. Physiol. Chem., 263, 175 (1940). (9) H. Borsook and J. W. Dubnoff, J. Biol. Chem., 81,629 (1947); the structure of acylguanidines is discussed by K. Matsumoto and H. Rapoport, J. Org. Chem., 33, 552 (1968). (10) V. M. Clark and A. R . Macrae, unpublished reults. ( 1 1) V. M. Clark and S. G. Warren, Nature (London), 199, 657 (1963). (12) A. Lapidot and D. Samuel, Biochim. Biophys. Acta. 111, 537 (1965). (13) S. Olezza, R. Moratti, and L. Speranza, Farmaco, Ed. Prat., 21,319 (1966); Chem. Abstf., 65, 7525h (1951). (14) A. A. Noyes, Z. Phys. Chem., 11, 495 (1893). (15) 0. Meyerhof and K. Lohmann, Biochem. Z.,196, 49 (1928). (16) 0. Folin, J. Biol. Chem., 17, 463 (1914); 0. Folin and H. Wu. ibid., 38, 81 (1919). (17) S. P. Dana and A. K. Grzybowski, J. Chem. Soc., 6004 (1963), 187 (1964). (18) M. J. Jorgenson and D. R. Hartter, J. Am. Chem. SOC.,85, 878 (1963). (19) T. C. Bruice and S. J. Benkovic, "Bioorganic Mechanisms", Vol. I, W. A. Benjamin, New York, N.'?,, 1966, Chapter 1. (20) A. H. Ennor and H. Rosenberg, Biochem. J., 57, 203 (1954). (21) R. K. Morton, Nature(London), 172, 65 (1953). (22) J. F. Van Pilsum and B. Hiller, Arch. Biochem. Biophys., 85, 483 (1959). (23) The metaphosphate mechanism for creatine kinase appears to have been conceived independently by Allen and Haake on the basis of mechanistic s t u d i e ~ .by ~ , M. ~ Cohn and co-workers on the basis of magnetic resonance and binding [see M. Cohn and JReuben, Acc. Chem. Res., 4, 214 (1972), and T. L. James and M. Cohn. J. Biol. Chem., 249, 2599 (1974)], and by E. J. Milner-White and D. C. Watts, Biochem. J., 122, 727 (1971), on the basis of binding and inhibition studies although the latter authors argued more forcefully for a direct displacement mechanism. (24) Metaphosphate as a reactive intermediate appears to have been suggested first by W. W. Butcher and F. H. Westheimer, J. Am. Chem. SOC..77,2420 (1955), and by A. R. Todd, Proc. Natl. Acad. Sci. U.S.A., 45, 1389 (1959). (25) M. Eigen and L. Demaeyer in "Techniques in Organic Chemistry", Vol. Vlli (part II),S. L. Friess. E. S. Lewis, and A. Weissberger, Ed., Interscience, New York, N.Y., 1963, p 1031. (26) K. Gaede and R. Grittner. Naturwissenschaften, 89, 39 (1952). (27) V. Paulus, M. G. Choc, and G. W. Allen, unpublished results. (28) K. Bloch. R. Schoenheimer, and D. Rittenberg. J. Bo/. Chem., 138, 155 (1941); K. Blochand R . Schoenheimer. ibid., 131, 111 (1939). (29) V. C. Myers and M. S. Fine, J. Biol. Chem., 21, 583 (1915). (30) M. Carpenter, P.McCay, and R. Caputto, Proc. SOC.Exp. Biol. Med., 97, 205 (1958). (31) A. Marty, R. Nordmann. and J. Nordmann, Bull. SOC.Chem. Bid., 43,619 (1961). (32) R. J. Block, E. L. Durrum, and G. Zweig, "Paper Chromatography and Paper Electrophoresis", Academic Press, New York, N.Y., 1958, p 348.
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